Engineering outer membrane vesicles (OMVs) carrying OmpA from S. Typhimurium for targeted modulation of human B-cell...

Salmonella enterica serovar Typhimurium is a Gram-negative bacterium recognized for its ability to invade and develop antimicrobial resistance. Among its virulence factors are outer membrane vesicles (OMVs), which play a significant role in modulating host immune responses by delivering components such as outer membrane proteins (OMPs). In this study, we engineered OMVs carrying OmpA (OmpA-OMVs) and investigated their effects on B-cell modulation. OMVs were isolated from Escherichia coli BL21(DE3) expressing recombinant OmpA and characterized using biophysical techniques. Furthermore, Raji human B-cells were stimulated with OmpA-OMVs, and their immunomodulatory effects were evaluated using RT-qPCR, western blotting, immunofluorescence, and chromatin immunoprecipitation assays. Importantly, OmpA-OMVs significantly upregulated Toll-like receptor 2 (TLR2) expression and activated NF-κB signaling in B-cells, leading to enhanced expression of activation-induced cytidine deaminase (AID), a key enzyme required for class switch recombination (CSR). This was accompanied by increased expression of AID transcriptional activators such as cMYC, PAX5, STAT6, and SMAD3 and decreased expression of the repressor cMYB. Chromatin immunoprecipitation analysis showed increased cMYC occupancy at the nuclear level over time. Functionally, OmpA-OMVs promoted IgA expression, indicating active isotype switching. These findings suggest that OmpA-OMVs enhance the immunogenic properties of OmpA and improve vesicular-mediated delivery, boosting immune activation compared to purified OmpA protein alone. Collectively, these findings support the potential use of OmpA-OMVs in targeted immunotherapies or vaccines against Salmonella infections.